Funct Integr Genomics (2014) 14:741–755 DOI 10.1007/s10142-014-0392-1

ORIGINAL PAPER

Linkage of cold acclimation and disease resistance through plant– pathogen interaction pathway in Vitis amurensis grapevine Jiao Wu & Yali Zhang & Ling Yin & Junjie Qu & Jiang Lu

Received: 27 December 2013 / Revised: 7 August 2014 / Accepted: 11 August 2014 / Published online: 26 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Low temperatures cause severe damage to none cold hardy grapevines. A preliminary survey with Solexa sequencing technology was used to analyze gene expression profiles of cold hardy Vitis amurensis ‘Zuoshan-1’ after cold acclimation at 4 °C for 48 h. A total of 16,750 and 18,068 putative genes were annotated for 4 °C-treated and control library, respectively. Among them, 393 genes were upregulated for at least 20-fold, while 69 genes were downregulated for at least 20-fold under the 4 °C treatment for 48 h. A subset of 101 genes from this survey was investigated further using reverse transcription polymerase chain reaction (RT-PCR). Genes associated with signaling events in pathogenassociated molecular pattern (PAMP)-triggered immunity (PTI), including generation of calcium signals (CNGC, CMLs), jasmonic acid signal (JAZ1), oxidative burst (Rboh), and phosphorylation (FLS2, BAK, MEKK1, MKKs) cascades, were upregulated after cold acclimation. Disease resistance genes (RPM1, RPS5, RIN4, PBS1) in the process of effectortriggered immunity (ETI) were also upregulated in the current condition. Defense-related genes (WRKYs, PR1, MIN7) involved in both PTI and ETI processes were abundantly expressed after cold acclimation. Our results indicated that plant–pathogen interaction pathways were linked to the cold acclimation in V. amurensis grapevine. Other biotic- and abiotic-related genes, such as defense (protein phosphatase 2C, U-box domain proteins, NCED1, stilbene synthase), transcription (DREBs, MYBs, ERFs, ZFPs), signal transduction (kinase, calcium, and auxin signaling), transport (ATPElectronic supplementary material The online version of this article (doi:10.1007/s10142-014-0392-1) contains supplementary material, which is available to authorized users. J. Wu : Y. Zhang : L. Yin : J. Qu : J. Lu (*) Viticulture and Enology Program, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China e-mail: [email protected]

binding cassette (ABC) transporters, auxin:hydrogen symporter), and various metabolism, were also abundantly expressed in the cold acclimation of V. Amurensis ‘Zuoshan1’ grapevine. This study revealed a series of critical genes and pathways to delineate important biological processes affected by low temperature in ‘Zuoshan-1’. Keywords Grape . Vitis amurensis . Cold tolerance . Gene expression . Real-time RT-PCR

Introduction Low temperatures include nonfreezing (2–6 °C) and freezing (0 °C and below) ones. The latter is a major environmental factor that causes dehydration to the plant cell and results in severe losses of production. During the process of freezing, ice forms first in the extracellular compartment of plant tissues, causing reduction of its water potential and leading to water loss in the cells through osmosis (Shinozaki and Yamaguchi-Shinozaki 2000). Plants, as sessile organisms, can achieve cold tolerance and survive freezing temperatures by exposing to the prior nonfreezing temperatures and adjusting to fit the subsequent much lower temperature condition. This evolved process is known as cold acclimation, a complex plant physiological and biochemical change involving growth morphology (Saltveit 2000), membrane structure, and cytoskeleton (Sangwan et al. 2001); accumulation of soluble proteins and antioxidants (Kang and Saltveit 2001); and increases in the activity of oxygen-scavenging enzymes (Brüggemann et al. 1999), sugar, proline, anthocyanin, and abscisic acid (ABA) content (Ait Barka and Audran 1997; Kumar et al. 2008; Miguel et al. 2004; Wanner and Junttila 1999). In addition, some cold-tolerant plants can produce specific proteins to protect cells under cold condition by lowering the freezing point (Antikainen and Griffith 1997;

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Yoshimune et al. 2005). Since the cold acclimation is very complex and is involved in almost every cellular process, it brings great challenge to figure out the critical process that may regulate cold tolerance in plant. Considerable progress has been made in understanding the mechanisms of cold tolerance triggered by cold acclimation in Arabidopsis and other plants, such as tomato (Zhang et al. 2004) and rice (Kim et al. 2010; Yun et al. 2010). The metabolite composition during the freezing tolerance has been predicted in Arabidopsis (Cook et al. 2004; Kaplan et al. 2004; Korn et al. 2010), and many cold tolerance genes, such as COR, KIN, LTI, RD, CBFs, and ICE1, have been identified (Zhu et al. 2007). Among them, the best known mechanism with a role in cold acclimation is the C-repeat binding factor (CBF) cold-responding pathway (Thomashow 2001). This cold tolerance process includes action of three transcription factors, CBF1, 2, and 3 (Gilmour et al. 1998; Jaglo et al. 2001; Medina et al. 1999). These transcription factors are rapidly induced in response to low temperature followed by expression of the CBF-targeted genes (CBF regulon). CBF1 and CBF3 are negatively regulated by CBF2 and have a concerted additive effect to induce the whole CBF regulon and the complete development of cold acclimation (Maruyama et al. 2009; Novillo et al. 2004, 2007). However, CBF does not appear to be the sole pathway conferring to freezing tolerance (Fowler and Thomashow 2002). For example, two transcription factors identified in Arabidopsis, Hos9 and Hos10 (high expression of osmotically responsive genes), are required for basal freezing tolerance but not regulated by CBF (Zhu et al. 2004, 2005). Furthermore, eskimo1 mutant of Arabidopsis described by Xin and Browse (1998) and ada2 mutants (ADA2 encodes a transcriptional adaptor protein) (Vlachonasios et al. 2003) are constitutively more freezing tolerant than wild-type plants, but they have different pathways distinct from CBF. Vitis vinifera grapevines, as one of the major fruit crops in the world, are relatively sensitive to cold temperature (Fuller and Telli 1999), although some other Vitis species such as Vitis amurensis and Vitis riparia are relatively cold hardy. Vitis species are deciduous woody perennials and have unique characteristics which are not able to be documented by the model species like Arabidopsis. Under field conditions, grapevines, like other woody perennials, develop deep winter hardiness in response to shortening day length and low temperatures (Weiser 1970). Shortening day length first initiates the transition from active growth to winter dormancy which results in a moderate increase in freezing tolerance, but subsequent exposure to low nonfreezing temperatures is required to promote the deep winter hardiness that is unique to woody perennials (Fuchigami et al. 1971; Weiser 1970). Also the woody perennials, like herbaceous species, can acquire cold tolerance when exposed to low temperatures during long days (Christersson 1978; Li et al. 2003). The mechanisms of cold

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tolerance induced by cold acclimation in woody perennials have been described on many species, such as eucalyptus (Navarro et al. 2009), pine (Beck et al. 2004), poplar (Benedict et al. 2006; Cocozza et al. 2009; Welling et al. 2002), citrus ( ahin-Çevik and Moore 2006), and peach (Tittarelli et al. 2009). All of which suggested that CBF pathway existed in the woody perennials. Vitis has become a new model plant for performing whole genome-wide study for fruit crops trees as the complete genome sequence of V. vinifera cv. Pinot Noir grape was released about 6 years ago (http://www.cns.fr/spip/Vitis-niniferae. html). Recently, gene expression levels have been detected in grape during the cold acclimation using transcript profiling method (Mathiason et al. 2009; Tattersall et al. 2007; Xin et al. 2013). Several proteins associated with cold tolerance have been isolated from grape, such as CBF1-4 (Xiao et al. 2006, 2008), H + -pyrophosphatase (Venter et al. 2006), and dehydrins (Xiao and Nassuth 2006). In the present study, ‘Zuoshan-1’, a clonal selection from wild V. amurensis with cold hardiness as low as −40~−50 °C, was employed to predict the frame of cold response in the acclimation of low temperature.

Material and methods Plant treatment and RNA extraction One-year-old, certified virus-free V. amurensis ‘Zuoshan-1’ seedlings (important Chinese grape cultivation collected from Changbai Mountain in Jilin Province in China) were maintained in the greenhouse under a 16-h light/8-h dark photoperiod at 25 °C, 85 % relative humidity. The well-grown plants were transferred from the greenhouse to a controlled growth chamber for weeks under a 16-h light/8-h dark photoperiod at 25 °C. Control plants were maintained under the same conditions. For cold treatment, plants were transferred to another growth chamber equipped with low temperature control and maintained at 4 °C under a 16-h light/8-h dark photoperiod. The fourth unfolded leaf from the main shoot apex was harvested from each of three vines, and the three leaves were combined to represent one biological replicate. Three independent biological replicates were collected. For Solexa sequencing, samples from three biological replicates were harvested at 48-h cold treatment and pooled for library construction. For real-time reverse transcription polymerase chain reaction (RT-PCR) experiment, another batch of seedlings was cold-treated for 2, 4, 8, 12, 24, and 48 h and harvested individually with three independent biological replicates. Total RNA was isolated from the sample using a modification of the CTAB method described by Murray and Thompson (1980) and purified with Qiagen’s RNeasy kit. The

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RNA quality was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

multiple test (Benjamini and Hochberg 1995). Pathways with Q value50 tags) in 4 °C library, 393 genes were upregulated and 69 DEGs downregulated for at least 20-fold in 4 °C library. These DEGs were classified into six categories including defense, transport, signal transduction, transcription, metabolism, and other functions (Additional File 3). Fig. 1 Comparison of gene expression level between the two libraries. For comparing gene expression level between ‘Zuoshan-1’ 4 °C (ZS1-4C) and control (ZS1-CON) library, each library was normalized to one million tags. Red dots represent transcripts more prevalent in 4 °C library, green dots show those present at a lower frequency in the 4 °C-treated tissue, and blue dots indicate transcripts that did not change significantly. The parameters “FDR≤0.001” and “log2 Ratio≥1” were used as the threshold to judge the significance of gene expression difference

Cold-regulated pathways deduced from the cold-responding genes All the differentially expressed genes were analyzed with pathway enrichment to have a preliminary full-field vision for the important biological processes. A total of 112 pathways were enriched with upregulated DEGs and 99 enriched with downregulated DEGs, respectively (Additional File 4). There were one significantly enriched pathway (Q value< 0.05) for upregulated DEGs and 10 for downregulated DEGs (Table 2). The only significantly enriched upregulated pathway is the plant–pathogen interaction pathway (pathway ID: ko04626) (http://www.kegg.jp/kegg-bin/highlight_pathway? scale=1.0&map=map04626&keyword=plant-pathogen interaction) (Fig. 2). The significantly enriched pathways for the downregulated DEGs are involved in metabolism (zeatin biosynthesis, porphyrin and chlorophyll metabolism, limonene and pinene degradation, stilbenoid/ diarylheptanoid/gingerol biosynthesis, glycosphingolipid biosynthesis, anthocyanin biosynthesis, photosynthesis, diterpenoid biosynthesis) and environmental adaptation (circadian rhythm—plant) (Table 2). DEGs by real-time RT-PCR A subset of 101 DEGs from the RNAseq data, including 96 upregulated and 5 downregulated genes, was investigated further by real-time RT-PCR (Additional File 1). Vitis EFα

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Table 2 List of the significantly enriched pathways for up- and downregulated EDGs Pathway

Pathways for upregulated DEGs Plant–pathogen interaction Pathways for downregulated DEGs Zeatin biosynthesis Porphyrin and chlorophyll metabolism Limonene and pinene degradation Stilbenoid, diarylheptanoid, and gingerol biosynthesis Circadian rhythm—plant Glycosphingolipid biosynthesis—ganglio series Biosynthesis of secondary metabolites Anthocyanin biosynthesis Photosynthesis Diterpenoid biosynthesis

DEGs tested (2,797)

All KEGG genes (109,020)

P value

Q value

Pathway ID

333

5,045

3.707591e-77

1.11163E-32

ko04626

9

372

7.879207e-05

0.007800415

ko00908

12 14 15 15 3 107 3 26 7

826 1,122 1,336 1,369 67 17,882 70 3,156 438

0.0006406261 0.001028101 0.001933711 0.002432571 0.003947702 0.004391182 0.004464443 0.00462057 0.005031538

0.031710992 0.033927333 0.047859347 0.048164906 0.049812226 0.049812226 0.049812226 0.049812226 0.049812226

ko00860 ko00903 ko00945 ko04712 ko00604 ko01110 ko00942 ko00195 ko00904

(forward primer 5′-TCCAAGGCCAGGTATGATG-3′; reverse primer 5′-CAGAGATTGGAACGAAGGG-3′) was used as reference gene for data normalization. All but eight genes were amplified in the real-time RT-PCR experiment. Functional analysis revealed that 32 genes were involved in plant–pathogen interaction pathway (Additional File 1 and Fig. 3), and the remainder were assigned to other pathways, including defense, transport, signal transduction, transcription, and metabolism (Additional File 1 and Fig. 4). Among all the tested DEGs, the group of top 10 genes that showed the largest response in real-time RT-PCR, including those associated with disease resistance, abiotic stress, signaling, and metabolism, is listed in Table 3. Pathogenesis-related protein 1, mitogen-activated protein kinase kinase 5, and JAZ1 involved in plant–pathogen interaction pathway showed dramatic response under cold stress. Stress-related genes, 9-cisepoxycarotenoid dioxygenase (NCED1), and ethyleneresponsive transcription factor ERF098-like were high expressed. Plant hormone-related genes (gibberellin 2-betadioxygenase 1, small auxin upregulated RNA (SAUR) family protein) and metabolite-associated genes (glutathione S-transferase parA, 4-coumarate–CoA ligase-like 7, serine acetyltransferase 3) were also abundantly expressed after cold acclimation in ‘Zuoshan-1’ (Table 3).

Cold acclimation activating plant–pathogen interaction pathway The preliminary RNAseq data depicted that plant–pathogen interaction pathway significantly responded to cold acclimation. In order to verify the involvement of plant–pathogen interaction pathway in cold tolerance of V. amurensis, 38 highly expressed genes in this pathway were verified by

real-time RT-PCR (Additional File 1 and Fig. 3). All but six genes were amplified in the real-time RT-PCR experiment. It has been well described the plant defense against invading pathogens is firstly triggered by pathogen-associated molecular patterns (PAMPs). The PAMPs are recognized by pattern recognition receptors (PRRs), subsequently leading to the PAMP-triggered immunity (PTI). In our study, a PRR that is also transmembrane chitin-binding protein, CEBiP (GSVIVT01023936001), was identified to be cold induced in RT-PCR experiment. During PTI process, various signaling events are triggered, including generation of calcium signals, oxidative burst, NO, and phosphorylation cascades. These signaling-associated genes were further verified by real-time RT-PCR. Among them, calcium signaling-associated genes cyclic nucleotide-gated channel (CNGC, GSVIVT01025696001) and calmodulin-like proteins (CMLs, GSVIVT01010382001, GSVIVT01013467001) were upregulated for 4-, 2-, and 17-fold, respectively. Rboh (GSVIVT01031128001), functioning as NADPH oxidase to generate reactive oxygen species (ROS), was expressed 9-fold higher in 48-h cold treatment. Moreover, genes involved in kinase cascades were upregulated after 48-h cold acclimation. For example, flagellin-sensitive 2 (FLS2, (GSVIVT01013974001), BRI1-associated receptor kinase 1 (BAK1, GSVIVT01014531001), MEK kinase 1 (MEKK1, GSVIVT01024709001), and MAP kinase kinases (MKKs, GSVIVT01015155001, GSVIVT01008476001) were abundantly expressed and upregulated for 3-, 6-, 6-, 3-, and 145fold, respectively, in the cold condition. JA signalingassociated gene JAZ1 (jasmonate ZIM-domain protein 1, GSVIVT01015042001) was 53-fold higher after the cold treatment. These signaling molecules trigger immunity response such as hypersensitive response (HR), cell wall reinforcement, stomatal closure, phytoalexin accumulation, and

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Fig. 2 Plant–pathogen interaction pathway. Proteins in red frame are encoded by upregulated DEGs in 4 °C library

defense-related gene induction which are pivotal to the plant resistance against different pathogens. Fig. 3 Real-time RT-PCR analysis for differentially expressed genes in plant–pathogen interaction pathway. Real-time RT-PCR analysis was conducted for analyzing genes in plant–pathogen interaction pathway in control (white) and 4 °C-treated (black) samples. All values were normalized to the Vitis EFα expression level. Data represent fold change of relative quantification of tested genes. Bars represent standard deviation calculated from three biological replicates

Pathogens have evolved effector proteins to suppress PTI, while plants have evolved resistance (R) proteins to recognize

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Fig. 4 Real-time RT-PCR analysis for differentially expressed genes in non-plant–pathogen interaction pathways. Real-time RT-PCR analysis was conducted for analyzing genes in various pathways except for the plant–pathogen interaction pathway. Control samples were shown in

white and 4 °C-treated samples in black. All values were normalized to the Vitis EFα expression level. Data represent fold change of relative quantification of tested genes. Bars represent standard deviation calculated from three biological replicates

these effectors accordingly which leads to next level of plant defense that is known as the effector-triggered immunity (ETI). Among those genes, encoding R proteins in ETI process, RPM1 (GSVIVT01007143001, GSVIVT01001444001), RPS5 (GSVIVT01034429001), RPM1 interacting protein 4 (RIN4, GSVIVT01020994001), and AvrPphB susceptible 1 (PBS1, GSVIVT01016915001, GSVIVT01015077001, GSVIVT01014538001) were also found being upregulated after the cold acclimation in ‘Zuoshan-1’. An effector target protein MIN7 (GSVIVT01023854001), involved in callose deposition for basal resistance of Arabidopsis to Pseudomonas syringae (Nomura et al. 2006), was upregulated for 6-fold after the cold treatment. In the plant–pathogen interaction pathway, PTI and ETI lead to induction of defense-related genes, including WRKYs, FRKs (receptor-like protein kinases), NHO1 (glycerol kinase), and pathogenesis-related proteins (PRs). Among these

defense-related genes, NHO1 (nonhost resistance gene 1, GSVIVT01015682001) was not detected, and WRKYs (GSVIVT01008553001, GSVIVT01033194001) and pathogenesis-related protein 1 (PR1, GSVIVT01037014001) genes were upregulated in the real-time RT-PCR analysis. Among these tested genes from plant–pathogen interaction p a t h w a y, t h r e e g e n e s ( G S V I V T 0 1 0 3 7 0 1 4 0 0 1 , GSVIVT01015042001, GSVIVT01027069001) were selected for the expression analysis using real-time RT-PCR at different time courses during cold acclimation (Fig. 5). GSVIVT01037014001 encoding PR1 started to increase soon after the cold treatment and reached 473-fold higher at 48 h after cold acclimation. GSVIVT01015042001 encoding JAZ1 showed similar expression trend with GSVIVT01037014001 and reached a peak of 53-fold at 48 h. Expression levels of the gene GSVIVT01027069001 encoding WRKY 30 gradually increased after cold treatment, reached a peak at 12 h (18fold), and then decreased to 10-fold at 48-h cold acclimation.

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Other abundantly expressed genes during cold acclimation Genes associated with biotic and abiotic stresses Stress-related genes, such as protein phosphatase 2C (GSVIVT01 034176001, 10-fold), U-box domain-containing protein (GSVIVT01020229001, 8-fold), NCED1 (GSVIVT010380 80001, 431-fold), and stilbene synthase (GSVIVT0 1010561001, 11-fold), were detected by real-time RT-PCR, and they showed induction at 4 °C treatment. Genes associated with transport ATP-binding cassette (ABC) transporter gene (GSVIVT01037789001), as a membrane transport-associated gene, was upregulated for 7-fold after cold treatment. Another transport-related gene auxin:hydrogen symporter (GSVIVT01026429001), involved in auxin polar transport, was found to be upregulated for 11-fold after the cold acclimation in ‘Zuoshan-1’ leaves. Genes associated with signaling It is notable that a bunch of calcium signaling genes were induced at the present condition, including calcium-binding protein (GSVIVT01020549001, 2fold), calmodulin-binding protein (GSVIVT01008322001, 34fold), and calmodulin-like protein (GSVIVT01018070001, 2fold). Some others encoding auxin-related signaling genes including auxin-response factor (GSVIVT01021552001, 25fold), auxin-induced protein (gi|110369896, 3-fold), auxinrepressed protein (gi|110404608, 20-fold), and SAUR family protein (GSVIVT01016698001, 138-fold) were also upregulated during cold acclimation in the RT experiment. Genes associated with transcription In the present study, three AP2/ERF family genes (GSVIVT01010631001, 1-fold; GSVIVT01028050001, 2-fold; GSVIVT01009007001, 2-

fold) were identified to be cold upregulated in the RT experiment. Two myb-related proteins (GSVIVT01028328001, 10fold; GSVIVT01015264001, 8-fold) and one ERF gene (GSVIVT01028314001, 338-fold) were induced by cold stress. WRKY family genes (e.g., GSVIVT01008553001, 9fold; GSVIVT01033194001, 2-fold; GSVIVT01019511001, 3-fold; GSVIVT01027069001, 10-fold), zinc finger proteins (GSVIVT01003473001, 30-fold; GSVIVT01033017001, 43fold), and NAC transcription factors (e.g., GSVIVT01038666001, 10-fold; GSVIVT01023921001, 1fold) were induced by cold stress in ‘Zuoshan-1’ leaves. Other transcription factors, such as bHLH104-like, jumonji (jmjC) domain-containing protein, ocs element-binding factor, and TCP8-like transcription factor, were also induced in ‘Zuoshan-1’ leaves after cold treatment based on the RT result. Genes associated with metabolism Many kinds of metabolism processes were induced among this category, including carbohydrate, protein, amino acid, secondary, energy and hormone metabolism, etc. A gene encoding beta-amylase 3 associated with carbohydrate metabolism was upregulated for 33-fold by cold treatment in this study. Protein metabolism also changed significantly after cold stress. These changes occurred in protein synthesis and degradation. Among these induced genes, RING finger proteins (e.g., gi|254914834, 4-fold) and cysteine protease family-related metacaspase (GSVIVT01013841001, 2-fold) were identified. Some proteases with proteolytic activity, such as ATP-dependent zinc metalloprotease (gi|254916896, 2-fold) and subtilisin-like protease (GSVIVT01006968001, 34-fold), were upregulated. The free amino acid contents also increased in ‘Zuoshan-1’ during cold treatment. A gene encoding serine acetyltransferase

Table 3 Top 10 genes that showed the largest response in real-time RT-PCR Gene

Description

Function

GSVIVT01038047001 ref|XP_002264703.1|PREDICTED:probable glutathione S-transferase parA [Vitis vinifera]

1,409

GSVIVT01037014001 GSVIVT01038080001

473 431

GSVIVT01003478001 GSVIVT01028314001 GSVIVT01000687001 gi|71868473 GSVIVT01008476001 GSVIVT01016698001 GSVIVT01015042001

Amino acid metabolism gb|ADN43428.1|pathogenesis-related protein 1 [Vitis shuttleworthii] Disease resistance ref|XP_002277354.1|PREDICTED:9-cis-epoxycarotenoid dioxygenase NCED1, chloroplastic Abiotic stress [Vitis vinifera] ref|XP_002270544.2|PREDICTED:serine acetyltransferase 3, mitochondrial-like [Vitis Amino acid vinifera] metabolism ref|XP_002264390.2|PREDICTED:ethylene-responsive transcription factor ERF098-like [Vitis Transcription vinifera] ref|XP_002263961.1|PREDICTED:gibberellin 2-beta-dioxygenase 1 [Vitis vinifera] Hormone metabolism ref|XP_002276353.1|PREDICTED:4-coumarate–CoA ligase-like 7 [Vitis vinifera] Secondary metabolism ref|XP_002283080.1|PREDICTED:mitogen-activated protein kinase kinase 5 [Vitis vinifera] Disease resistance ref|XP_002326308.1|SAUR family protein [Populus trichocarpa] Signaling gb|AEP60132.1| JAZ1 [Vitis rupestris] Disease resistance

RT fold

419 338 168 155 145 138 53

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Fig. 5 Real-time RT-PCR based expression analysis of three genes in plant–pathogen interaction pathway. The relative expression levels of three genes (GSVIVT01037014001, GSVIVT01015042001, GSVIVT01027069001) were analyzed by real-time RT-PCR after 4 °C treatment for 0, 2, 4, 8, 12, 24, and 48 h. All values were normalized to the

expression level of the Vitis EFα gene. Data represent the “fold change” in gene expression in 4 °C-treated vs. untreated leaf samples. Values represent relative levels of gene expression with bars of standard deviation from three biological replicates

(GSVIVT01003478001) was upregulated for 419-fold after cold treatment. Four genes encoding glutathione S-transferase (GST) (GSVIVT01038047001, 1,409-fold; gi|110430232, 27fold; GSVIVT01038031001, 4-fold; GSVIVT01019987001, 7-fold) were increased at low temperature. In addition, low temperature stimulated 1-aminocyclopropane-1-carboxylate (ACC) oxidase (gi|156723741, 5-fold) and phenylalanine ammonia-lyase (GSVIVT01024306001, 3-fold) activity in ‘Zuoshan-1’. Some others, such as serine acetyltransferase (GSVIVT01003478001, 419-fold), lysine-specific demethylase (GSVIVT01024382001, 1-fold), and tyrosine aminotransferase (GSVIVT01020585001, 2-fold), were also upregulated in our study. The subcategory of secondary metabolism, such as metabolites of phenols, organic acid, natural pigment, alkaloid, vitamin, monoterpene, etc., was accumulated after cold stress. In RT experiment, putative genes related to the synthesis of dihydroflavonol (GSVIVT01013135001, 4-fold) and isoflavone (GSVIVT01021135001, 7-fold) were upregulated. Some genes related to organic acid metabolism were upregulated, including caffeic acid 3-O-methyltransferase (GSVIVT01019691001, 1-fold), 4-coumarate–CoA ligaselike 7 (gi|71868473, 155-fold), and trans-cinnamate 4monooxygenase (GSVIVT01032256001, 16-fold). We found a group of upregulated genes regulating the synthesis of natural pigment, such as anthocyanidin. Some others involved in tropinone (GSVIVT01016487001, 6-fold), riboflavin (gi|22015079, 10-fold), and secologanin (GSVIVT01035466001, 2-fold) metabolism were also detected in our study. Genes belonging to energy metabolism were also enhanced after cold treatment. This group was mainly comprised of genes encoding nicotinamide adenine dinucleotide (NADH) dehydrogenase (GSVIVT01005840001, 19-fold), cytochrome P450 (GSVIVT01000764001, 2-fold), ferredoxin (gi|110376624, 12-fold), and ATP synthase (gi|312647766, 4-fold).

Plant hormones also have an important role during chilling stress. Gibberellin (GAs), cytokinin, indole-3-acetic acidamido (IAA), and ABA-related genes were induced in ‘Zuoshan-1’ leaves by cold treatment. Gibberellin 2-betadioxygenase (GSVIVT01000687001, 168-fold), which degrades active GAs and is involved in cold response (Thomas et al. 1999), was upregulated in our study. A cytokinin biosynthesis gene, cytokinin hydroxylase (GSVIVT01028930001, 5fold), was upregulated. After cold acclimation, IAA level may increase by the high expression of a gene encoding indole-3acetic acid-amido synthetase (GSVIVT01037892001, 9-fold). In addition, abscisic acid 8′-hydroxylase (GSVIVT01036885001, 20-fold) was highly expressed after cold treatment in the RT experiment.

Discussion The aim of the study was to determine a core set of coldresponding genes and conjecture the important pathways regulated by low temperature in V. amurensis cv. Zuoshan-1. A preliminary survey with the Solexa sequencing technology was used to analyze gene expression profiles of cold hardy V. amurensis cv. Zuoshan-1 after cold acclimation at 4 °C for 48 h. Xin et al. (2013) used the same transcriptomic sequencing technology to study gene expression of V. amurensis under the 8-h cold stress. However, there are several key differences between these two studies. For example, cultivars, tissues, and the time courses under cold treatment were different. While we used the fourth unfolded leaves from the main shoot apex of V. amurensis ‘Zuoshan-1’ that were under cold treatment for 48 h, their studies used 8-h cold-treated shoot apices of V. amurensis. When the expression data from these two experiments were compared, very different cold-regulated gene expressing patterns were observed. For example, in 8-h cold treatment, a total of 1,314 genes were changed at least 2-fold,

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while in our study, 5,230 DEGs were identified after 48-h cold acclimation. It suggests that V. amurensis may quickly respond to cold stress by altering a set of genes in the early stage of cold exposure. After an extended period of cold acclimation for 48 h, V. amurensis intensively mobilizes many more transcripts and overall viability to overcome the potential cold damage. A detailed comparison of the two studies suggests that V. amurensis can initiate cold stress tolerance process soon after exposure to cold stress by altering expression level of a number of genes, especially the transcription factors. While most of the genes detected in 8-h cold treatment were still highly active in 48 h of cold stress, other highly expressed genes appeared in 48 h after cold treatment indicating that many more biological processes, particularly those defensive-related systems, were mobilized to protect the grapevines from further damage. Cold cues sensing and transporting During cold acclimation, plants sense environmental cues and series of biochemical and physiological changes, building up protection from freezeinduced injury. Recent studies indicate that plant cell membranes may play a major role in sensing low temperatures. The membrane, especially the plasmalemma, is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. One ABC transporter gene (e.g., GSVIVT01037789001, 7-fold) was found to be upregulated during the cold acclimation in grape leaves. It may have complex function in polar auxin transport, secondary metabolite transport, lipid catabolism, xenobiotic detoxification, chlorophyll biosynthesis, Fe/S cluster formation, ion fluxes, disease resistance, stomatal movement, and cell elongation processes (Marin et al. 2006; Rea 2007; Yazaki 2006). The multiple function of this transporter may be involved in biochemical and physiological adaption of ‘Zuoshan-1’ to cold stress. Transduction of cold signals Perception by cell membranes results in transient increases in cytosolic Ca2+ levels, triggering the cascading reactions to enhance cold tolerance of plants (Lissarre et al. 2010). Signal transduction by Ca2+, kinases, and plant hormones plays important roles in triggering cold pathways. It is notable that a bunch of calcium signaling genes were induced during the cold acclimation of ‘Zuoshan-1’ grapevine. Calcium is known to play a very important role in cold-sensing pathway (Carpaneto et al. 2007; Doherty et al. 2009; Knight et al. 1991, 1996; Monroy and Dhindsa 1995; Nordin Henriksson and Trewavas 2003). Plants possess specific multigene families of protein kinases that play crucial roles in mediating calcium signaling. Components of kinase cascades are induced or activated by cold and other abiotic stresses to induce downstream gene expression and genetic modification (Pitzschke and Hirt 2006; Sanders et al. 2002). In our study, many kinase-associated genes were induced by cold

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acclimation. In addition, some genes encoding plant hormonerelated signaling genes were also upregulated during cold acclimation. For instance, we found GA-, IAA-, and ABArelated genes were induced in ‘Zuoshan-1’ leaves by cold treatment. A gene encoding gibberellin 2-beta-dioxygenase (GSVIVT01000687001) was dramatically upregulated for 168-fold after cold acclimation. Accumulation of gibberellin 2-beta-dioxygenase, which degrades active GAs and is involved in cold response (Thomas et al. 1999), implies that active GAs level may be reduced, leading to suppress the plant growth under cold stress. Similar to the transition from active growth to winter dormancy, the growth reduction may help plants to adapt the cold stress by balancing the overall viability. A gene encoding IAA synthetase was increased for 9-fold in ‘Zuoshan-1’ grapevine after cold treatment. The increase of IAA level may have resulted from the high expression of IAA synthetase. Another gene encoding auxin:hydrogen symporter was increased for 11-fold under cold stress. It has been reported to be involved in auxin polar transport which may regulate auxin-mediated signaling pathway (Petrášek and Friml 2009). Our results also showed that the abscisic acid 8′-hydroxylase, an intracellular messenger that could play an important role in improving plant tolerance to cold (Llorente et al. 2000; Xiong et al. 2001), expressed 20-fold higher after cold treatment. Although GA, IAA, and ABA signaling was enhanced after cold treatment, little is known about their exact roles under cold stress. Transcription factors respond to cold stress After transduction of cold signals, transcription factors (TFs) fast respond to accumulate the relevant transcripts to counter temperature damage. Many TFs, such as DREBs (Xiao et al. 2006, 2008), AP2/ERFs (Haake et al. 2002; Tillett et al. 2012), myb-related proteins (Agarwal et al. 2006; Zhu et al. 2005), NACs (Collinge and Boller 2001; Hegedus et al. 2003; Nogueira et al. 2005), WRKYs (Eulgem et al. 2000; Mare et al. 2004; Talanova et al. 2009), and zinc finger proteins (Kim et al. 2001; Liu et al. 2007; Mukhopadhyay et al. 2004) have been reported to be regulated by biotic and abiotic stress, including extreme low temperature stress in various plants. Similar to those previous reports, the TFs were also upregulated in our study. Except these cold-regulated TFs, some other TFs such as bHLH104-like, jumonji (jmjC) domaincontaining protein, ocs element-binding factor, and TCP8like transcription factor were also induced in ‘Zuoshan-1’ grapevines after cold treatment. Since they have not been reported to regulate cold tolerance in plants, further investigation of these TFs will be necessary in order to better understand the role of these transcription factors on cold hardiness of grapevine. Expression of cold-responding defense proteins We found plant–pathogen interaction pathway was activated by the cold

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treatment in ‘Zuoshan-1’. Among them, genes encoding CDPKs (Monroy et al. 1993), NOS (Zhao et al. 2009), MEKK (Jonak et al. 1996), JAZ (Van Dam 2009), MYC2 (Chen et al. 2012), and WRKY (Talanova et al. 2009) have been found to play important roles in both biotic and abiotic stresses. In addition, PR protein genes, known as systematic response to pathogen attack, were clearly triggered by the low temperature in the present study. Induction of PR proteins by low temperature has already been shown in many plant species such as rye (Hon et al. 1994), bermuda grass (Gatschet et al. 1996), and potato (Zhu et al. 1993). These studies demonstrated that PR proteins such as endo-chitinase (Yeh et al. 2000), ß-1,3glucanase (Pihakaski-Maunsbach et al. 2001), glucanase-like (GLPs), chitinase-like (CLPs), and thaumatin-like proteins (TLPs) (Hon et al. 1995) were enriched in plants exposed to low hardening temperature. The role of these PR proteins in cold tolerance may be similar to antifreeze proteins (AFPs) (Hincha et al. 1997; Hon et al. 1995; Pihakaski-Maunsbach et al. 1996; Pihakaski-Maunsbach et al. 2001). They accumulated in plant cells to protect intracellular proteins and membrane during the freeze–thaw process (Anchordoguy et al. 1987). Many genes involved in PTI and ETI processes are upregulated after pathogen infection, leading to trigger immunity response such as HR, cell wall reinforcement, stomatal closure, phytoalexin accumulation, and defense-related genes induction which are pivotal to the plant resistance against different pathogens. For example, GSVIVT01037014001 is a PR1 protein with defensive function against pathogen in plants (Alexander et al. 1993; Li et al. 2011; Sarowar et al. 2005). GSVIVT01015042001 encodes JAZ1, degradation of which through the ubiquitin–proteasome pathway activates the expression of JA-responsive genes in the plant–pathogen interaction pathway (Hamel et al. 2011). GSVIVT01027069001 encodes WRKY transcription factor 30, which is regarded as critical regulatory component of plant responses to pathogen infection (Peng et al. 2012). In this study, they were selected for the expression analysis at different time courses during cold acclimation, and accumulation of these genes was detected soon after the cold treatment and held at relatively high level during the entire cold stress. Even though their function on cold resistance was largely unknown, it is inferred that they are positively involved in the cold tolerance of V. amurensis. Except the defense genes in plant–pathogen interaction pathway, defense genes in other pathways also showed induction at 4 °C treatment. For example, protein phosphatase 2C (Widjaja et al. 2010; Hu et al. 2010), 9-cis-epoxycarotenoid dioxygenase (Xian et al. 2014; Fan et al. 2009), and stilbene synthase (Xu et al. 2010; Dai et al. 2012) have been found to be involved in pathogen- and coldinducible stresses.

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Expression of cold-responding metabolites Various metabolism processes occurred after cold treatment in ‘Zuoshan-1’. An important component of adaptation to low temperature stress is the accumulation of compatible carbohydrates which can stabilize the cellular osmotic pressure. It has been reported that galactose (Morsy et al. 2007), sucrose (Sowiński et al. 1999) and xyloglucan (Polisensky and Braam 1996) are more pronounced in cold-tolerant plants. It has been reported that a starch-degrading enzyme, beta-amylase 3, was induced at low temperature (Hill et al. 1996; Wegrzyn et al. 2000). The same enzyme was found to be upregulated by cold treatment in ‘Zhushan-1’ vine. In the present study, genes involved in protein metabolism were upregulated significantly after cold stress in ‘Zuoshan1’. Among these induced genes, RING finger proteins and cysteine protease family-related metacaspase have been reported to have regulatory role in cold stress (Dong et al. 2006; Gao et al. 2012; Liu et al. 2008; Watanabe and Lam 2011). Subtilisin-like protease was another protein upregulated, which has been well induced by pathogen (Tornero et al. 1997) and involved in the regulation of stomatal development (Berger and Altmann 2000; von Groll et al. 2002) and temperature adaptation (Sigurðardóttir et al. 2009). Genes associated with amino acid metabolism were also increased in ‘Zuoshan-1’ during cold treatment. For example, a gene encoding serine acetyltransferase was upregulated for 419-fold, which plays a regulatory role in the biosynthesis of cysteine by its property of feedback inhibition by cysteine in bacteria and certain plants (Noji et al. 1998). Genes encoding GST were increased at low temperature, which help to catalyze the scavenging of ROS and protect plants against toxic oxygen stress triggered by low temperature (Roxas et al. 2000). In addition, low temperature stimulated ACC oxidase and phenylalanine ammonia-lyase activity and paralleled the increase in ethylene production which may play signaling transduction role in cold stress (John 1997; Lafuente et al. 2001). The subcategory of secondary metabolism, such as metabolites of phenols, organic acid, natural pigment, alkaloid, vitamin, monoterpene, etc., were accumulated during cold stress. Many of these metabolites can act in defense mechanisms against low temperature. For example, phenolic compounds have been reported as an antioxidant to protect plants from damage caused by ROS in cold stress (Christie et al. 1994; Pennycooke et al. 2005). Genes related to the synthesis of dihydroflavonol and isoflavone were upregulated too, indicating the accumulation of these phenolic compounds may improve the cold resistance. Some upregulated genes were related to organic acid, another kind of very important secondary metabolite in cold stress (Cook et al. 2004). We also found a group of upregulated genes regulating the synthesis of natural pigment, such as anthocyanidin, which have been reported to be

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accumulated in cold acclimation (Krol and Huner 1985; Leyva et al. 1995). Genes belonging to energy metabolism were also enhanced during cold treatment, such as NADH dehydrogenase, cytochrome P450, ferredoxin, and ATP synthase. These patterns support the assumption that ‘Zuoshan-1’ leaves are more metabolically active and have more energy needs during 48h cold acclimation to withstand the lower temperature in winter.

Acknowledgments This work was supported by the Chinese Universities Scientific Fund (Grant No. 2012RC019) and earmarked fund for Modern Agro-industry Technology Research System (CARS-30-yz-2).

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